PROJECT SUMMARY Chemical modifications of DNA and histones represent carriers of regulatory information that cooperate with transcription factors to control genome accessibility. Methylation at histone H3 lysine 36 (H3K36) is an evolutionarily conserved chromatin modification. In yeast, a single methyltransferase Set2 catalyzes all methylation states on H3K36. In contrast, H3K36 methylation states are subject to more complex regulation in mammalian chromatin. Whereas SETD2 is the sole enzyme catalyzing tri-methyl H3K36 (H3K36me3), several additional enzymes including NSD family members and ASH1L have evolved as the methyltransferases specific for di-methyl H3K36 (H3K36me2). Accordingly, the genomic distribution of H3K36me2 is distinct from that of H3K36me3, implying a possible functional divergence that remains incompletely understood. Germline mutations in NSD and ASH1L lead to various neurodevelopmental disorders, whereas somatic alterations of these enzymes are frequently found in human cancers. Recent work by us and others have identified and elucidated recurrent mutations directly affecting histone H3K36 that induced global depletion of H3K36me2 in human bone and head and neck cancers. Therefore, precise regulation of NSD/ASH1L-mediated H3K36me2 is critical for human development and represents a key barrier to neoplastic transformation. Despite its importance, there is limited understanding of the regulatory mechanisms governing H3K36me2 establishment and its function in genome regulation. Therefore, the five-year goal of my research program is to systematically examine the chromatin biology of H3K36me2 and its associated modifiers. The conceptual innovation is built on our recent findings of an interplay between H3K36me2 and intergenic DNA methylation through the recruitment of de novo DNA methyltransferase DNMT3A. In addition, we have developed tractable experimental systems and novel CRISPR/Cas-based genome- and epigenome-editing platforms that allow us to dissect the regulation of H3K36me2 in a multiplexed manner. We will determine the respective contribution of NSD and ASH1L to establishing H3K36me2 at introns and intergenic regions. A combination of chromatin biochemistry and functional genetic screening approaches will be employed to understand the regulatory input that guides the formation of H3K36me2. We will perform structural-functional analysis to better understand the molecular basis of DNMT3A recruitment by H3K36me2, and kinetic studies to delineate its relationship with the well-known antagonism between H3K36me2 and H3K27 methylation. Lastly, we will employ integrative epigenomics and epigenome-editing tools to determine the impact of H3K36me2 on the accessibility of cis-regulatory and repetitive elements. Together, these studies will provide a knowledge base upon which mechanistic insights into pathogenesis and targets amenable for therapy can be uncovered for human diseases driven by H3K36me2 dysregulation. Furthermore, the technical tools and resources could be readily applied to the study of additional chromatin modifications and of broad interest to the chromatin research community.